The application of low energy collisionally activated decomposition/tandem mass spectrometry (MS) to the characterization of peptides is currently limited by imperfect understanding of the gas-phase chemistry of peptide ions. Our recent tandem MS analyses of tryptic peptides derived from human a B-100, together with synthetic analogues, have revealed unexpected fragmentation behavior of the protonated ions. Thus, oxidation of cysteine to cysteic acid in peptides containing a C-terminal arginine residue, markedly promotes C-terminal ("Y" series) fragment ions, yielding clear sequence information. The data are consistent with an intra-ionic interaction between amino acid side-chains, promoting a multiplicity of sites of charge in the [M + H]+ species. We have also investigated other aspects of the low energy decomposition of protonated and metal-cationized peptides. A C-terminal rearrangement of protonated peptides (with loss of the C-terminal amino acid residue but retention of a carboxyl oxygen) has been studied using [18O]-labelling and MS/MS/MS. [18O] isotope exchange in the gas phase has also been demonstrated. The propensities to C-terminal rearrangement and [18O] exchange are sequence dependent. In combination, our data suggest a hypothesis of broad analytical significance, namely that low energy fragmentations of protonated and metal cationized by the site or sites of charge location and by the conformation (in part induced by protonation or other cationization) adopted by the peptide ion in the gas phase. The proposed research will evaluate this hypothesis through analyses of selected oligopeptides using fast atom bombardment- and electrospray-tandem MS (including MS/MS/MS techniques such as sequential product ion scanning and reaction intermediate scanning). We will investigate the fragmentations of series of peptides which differ with respect to the proton (or metal cation) affinities of their constituent amino acids and with respect to conformation. Selected derivatization procedures will be evaluated for their effect on gas phase proton and metal cation affinities and on the fragmentations of oligopeptides. Our studies of synthetic peptides will guide analytical strategies for the characterization of tryptic fragments of modified apoprotein B-100 derived from oxidized human low density lipoprotein (LDL). Particular attention will be paid to the recognition of oxidized cysteine and methionine residues and to the characterization of modifications associated with interaction of the protein with oxidized lipid constituents of LDL. Thus, tryptic fragments incorporating conjugation with malondialdehyde and 4-hydroxynonenal will be selectively isolated by immunoaffinity extraction for characterization by tandem MS. The combination of fundamental and analytical studies proposed here addresses our basic premise that proficiency in peptide structural characterization using tandem MS is dependent on improved understanding of the gas-phase chemistry of peptide ions.